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High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

ABSTRACT: High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

PROVIDER: PRJNA600540 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10871700.fastq.gz	Fastqsanger.gz
	SRR10871701.fastq.gz	Fastqsanger.gz
	SRR10871702.fastq.gz	Fastqsanger.gz
	SRR10871703.fastq.gz	Fastqsanger.gz
	SRR10871704.fastq.gz	Fastqsanger.gz

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High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

Project description:Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including next-generation gene therapy. However, due to the limited understanding of context-dependent structure-function relationships, the identification of functional riboswitches requires large-scale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8x104 constructs) by cDNA-amplicon-sequencing in transiently transfected and stimulated human cells. The self-barcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches. DOI:10.1038/s41467-020-14491-x

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High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells.

Project description:Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including next-generation gene therapy. However, due to the limited understanding of context-dependent structure-function relationships, the identification of functional riboswitches requires large-scale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8 × 104 constructs) by cDNA-amplicon-sequencing in transiently transfected and stimulated human cells. The self-barcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches.

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